Toxicity Assessment of Environmental Liquid Crystal Monomers: A Bacteriological Investigation on Escherichia coli and Staphylococcus epidermidis.

The widespread existence of liquid crystal monomers (LCMs) in various environmental matrices has been demonstrated, yet studies on the toxicological effects of LCMs are considerably scarce and are urgently needed to be conducted to assess the adverse impacts on ecology and human health. Here, we conducted a bacteriological study on two representative human commensal bacteria, Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis), to investigate the effect of LCMs at human-relevant dosage and maximum environmental concentration on growth, metabolome, enzymatic activity, and mRNA expression. Microbial growth results exhibited that the highest inhibition ratio of LCMs on S. epidermidis reached 33.6% in our set concentration range, while the corresponding data on E. coli was only 14.3%. Additionally, LCMs showed more dose-dependent toxicity to S. epidermidis rather than E. coli. A novel in vivo solid-phase microextraction (SPME) fiber was applied to capture the in vivo metabolites of microorganisms. In vivo metabolomic analyses revealed that dysregulated fatty acid metabolism-related products of both bacteria accounted for >50% of the total number of differential substances, and the results also showed the species-specific and concentration-dependent metabolic dysregulation in LCM-exposed bacteria. The determination of enzymatic activity and mRNA relative expression levels related to oxidative stress confirmed our speculation that the adverse effects were related to the oxidative metabolism of fatty acids. This study complements the gaps in toxicity data for LCMs against bacteria and provides a new and important insight regarding metabolic dysregulation induced by environmental LCMs in human commensal bacteria.

The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila.

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.

A novel virus-induced cyclic dinucleotide, 2'3'-c-di-GMP, mediates STING-dependent antiviral immunity in Drosophila.

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP. This CDN binds to and activates the protein STING to trigger immunity. We recently discovered in the model organism Drosophila melanogaster two cGAS-like receptors (cGLRs) that activate STING-dependent antiviral immunity and can produce 3'2'-cGAMP, in addition to 2'3'-cGAMP. Here we explore CDN-mediated immunity in 14 different Drosophila species covering 50 million years of evolution and report that 2'3'-cGAMP and 3'2'-cGAMP fail to control infection by Drosophila C virus in D. serrata, D. sechellia and D. mojavensis . Using an accurate and sensitive mass spectrometry method, we discover an unexpected diversity of CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster , including a novel CDN, 2'3'-c-di-GMP. We show that 2'3'-c-di-GMP is the most potent STING agonist identified so far in D. melanogaster and that this molecule also activates a strong antiviral transcriptional response in D. serrata . Our results shed light on the evolution of cGLRs in flies and provide a basis for the understanding of the function and regulation of this emerging family of PRRs in animal innate immunity.

Tetrazolyl Porphyrin-Based Hydrogen-Bonded Organic Frameworks: Active Sites-Mediated Host-Guest Synergy for Advanced Antimicrobial Applications.

Hydrogen-bonded organic frameworks (HOFs) with multiple functions and permanent pores have received widespread attention due to their potential applications in gas adsorption/separation, drug delivery, photocatalysis, proton conduction, and other fields. Herein, we constructed a three-dimensional (3D) HOF with 1D square channels by utilizing a dual-functional tetrazolyl porphyrin ligand bearing an active center of the porphyrin core and open sites of nitrogen atoms through π-π stacking and hydrogen-bonding interaction self-assembly. The structure exhibits both solvent resistance and thermal stability, and especially, maintains these after being transformed into nanoparticles. Meanwhile, the active sites exposed on the inner wall of the pores can interact well with the photoactive cationic dye molecules to form an effective host-guest (H-G) system, which can realize boosted photosensitized singlet oxygen (1O2) production under red light irradiation and synergistic sterilization toward Staphylococcus aureus (S. aureus) with an inhibition ratio as high as 99.9%. This work provides a valuable design concept for HOF-related systems in pursuit of promoted photoactivity.

In vivo environmental metabolomic profiling via a novel microextraction fiber unravels sublethal effects of environmental norfloxacin in gut bacteria.

Emerging contaminants (ECs), especially antibiotics, have significantly polluted the environment and threaten the living circumstance of organisms. Environmental metabolomic has emerged to investigate the sublethal effects of ECs. However, lacking noninvasive and real-time sample pretreatment techniques restricts its development in environmental toxicology. Hence, in this study, a real-time and in vivo untargeted analytical technique towards microbial endogenous metabolites was developed via a novel composite solid-phase microextraction (SPME) fiber of ZIF-67 and polystyrene to realize the high-coverage capture of living gut microbial metabolites. To reveal the exposure risks of typical antibiotic - norfloxacin (NFX) to gut bacteria, four representative bacteria were exposed to NFX at environmentally relevant levels. Using the proposed SPME fiber, 70 metabolites were identified to obtain an apparent metabolic separation feature between control and NFX-treated (10 ng/mL) microbial groups, which revealed that the low environmental relevant concentration of NFX would affect normal metabolism of gut bacteria. Additionally, NFX exhibited species-specific toxic effects on microbial growth, especially Escherichia coli displaying a distinct dose-dependent trend. Antioxidative enzymatic activities results demonstrated that beneficial bacteria maintained the state of oxidative stress while symbiotic bacteria suffered from oxidative stress injury under NFX contamination, further corroborating its impact on human intestinal health. This study highlights the suitability of in vivo SPME in the field of metabolite extraction and simultaneously possesses a brilliant application foreground in the environmental metabolomics.

Rapid and sensitive analysis of trace ß-blockers by magnetic solid-phase extraction coupled with Fourier transform ion cyclotron resonance mass spectrometry.

A rapid and sensitive method for analyzing trace ß-blockers in complex biological samples, which involved magnetic solid-phase extraction (MSPE) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), was developed. Novel nanosilver-functionalized magnetic nanoparticles with an interlayer of poly(3,4-dihydroxyphenylalanine) (polyDOPA@Ag-MNPs) were synthesized and used as MSPE adsorbents to extract trace ß-blockers from biological samples. After extraction, the analytes loaded on the polyDOPA@Ag-MNPs were desorbed using an organic solvent and analyzed by FTICR-MS. The method was rapid and sensitive, with a total detection procedure of less than 10 min as well as limits of detection and quantification in the ranges of 3.5-6.8 pg/mL and 11.7-22.8 pg/mL, respectively. The accuracy of the method was also desirable, with recoveries ranging from 80.9% to 91.0% following the detection of analytes in human blood samples. All the experimental results demonstrated that the developed MSPE-FTICR-MS method was suitable for the rapid and sensitive analysis of trace ß-blockers in complex biological samples.

Luteolin Suppresses Microglia Neuroinflammatory Responses and Relieves Inflammation-Induced Cognitive Impairments.

Microglia-mediated neuroinflammation in response to injurious self and non-self-stimuli exerts detrimental effects on neurons, which may lead to cognitive impairment. Luteolin, a typical kind of natural flavonoid in honeysuckle, chrysanthemum, and Herba Schizonepetae, is widely recognized to be anti-inflammatory and antioxidant against peripheral inflammation. However, its protective effect against inflammation-induced cognitive impairment is currently unknown. In this paper, we investigated the relief potential of luteolin against lipopolysaccharide (LPS)-induced cognitive impairment and neuroinflammation and its possible anti-inflammatory mechanisms in lipopolysaccharide-stimulated BV2 microglia cells. In this study, luteolin ameliorated LPS-induced cognitive impairments, indicated by behavioral performance of neuroinflammatory model mice in Morris water maze tests. Protein analyses and histological examination also revealed protective effect of luteolin against neuronal damage, through inhibiting overproduction of inflammatory cytokines in both hippocampus and cortex of mice. We also observed luteolin in vitro significantly suppressed the levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1 ß (IL-1ß), and inflammatory mediators like nitric oxide. Taken together, these results demonstrated luteolin was effective in alleviating cognitive impairment and limited neuronal damage via inhibiting the release of inflammatory mediators, suggesting luteolin is potential for further therapeutic research of neuroinflammation-related neurodegenerative diseases.

Recognition of pathogens in food matrixes based on the untargeted in vivo microbial metabolite profiling via a novel SPME/GC × GC-QTOFMS approach.

Foodborne diseases incurred by pathogenic bacteria are one of the major threats in food safety, and thus it is important to develop facile and effective recognition methodology of pathogens in food. Herein, a new automatic approach for detection of in vivo volatile metabolites emitted from foodborne pathogens was proposed by coupling solid phase microextraction (SPME) technique with a comprehensive two-dimensional gas chromatography quadrupole time-of-flight mass spectrometry (GC × GC-QTOFMS). A novel polymer composite based SPME probe which possessed high-coverage of microbial metabolites was utilized in this contribution to realize the sensitive extraction of untargeted metabolites. As a result, a total of 126 in vivo metabolites generated by the investigated pathogens were detected and identified, with 33, 29, 25, 21 and 18 volatile metabolites belonging to Shigella sonnei, Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus and Staphylococcus aureus, respectively. Multivariate statistical analyses were applied for further analysis of metabolic data and separation of responsive metabolic features among different microbial systems were found, which were also successfully verified in foodstuffs contaminated by microorganisms. The growth trend of the potential volatile markers of each pathogen in food samples kept consistent with that of the pure strain incubated in medium during the whole incubation time. This study promotes the application of SPME technology in microbial volatile metabolomics and contributes to the development of new approaches for foodborne pathogens recognition.

Combined effect of microplastics and DDT on microbial growth: A bacteriological and metabolomics investigation in Escherichia coli.

Microplastics (MPs) can adsorb toxic chemicals in biological or environmental matrixes and thus influence their behavior and availability. In order to investigate how the combined pollution of MPs and toxic organic chemical influence microbial growth and metabolism, Escherichia coli (E. coli) was grown in a complex, well-defined media and treated with polystyrene microplastics (PS MPs) and dichloro-diphenyl-tricgloroethane (DDT) at human relevant concentration levels. In vivo metabolites captured by a novel solid phase microextraction (SPME) probe, were used to reflect the metabolic dysregulation of E. coli under different pollution stresses. Results showed that the toxic effect of DDT displayed a distinct dose-dependent phenomenon while the existence of PS decreased the growth and metabolic interference effect of DDT on E. coli. Adsorption results revealed a mechanism that PS weakened the adverse impact of DDT by decreasing its free concentration in the treated culture media. Tricarboxylic acid (TCA) cycle related enzymes activities and antioxidant defense related substances of E. coli also proved the mechanism. The current study is believed to broaden our understanding of the ecotoxicity of MPs with toxic organic chemicals on microorganism.

In situ detection and imaging of lysophospholipids in zebrafish using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry.

In this paper, a matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) (MALDI-FTICR-MS) imaging method was developed to rapid and in situ detect the spatial distribution of lysophospholipids (LPLs) in zebrafish. The combination of MALDI with ultrahigh-resolution FTICR-MS achieves the MS imaging of LPLs with a mass resolution up to 50 000, which allows accurate identification and clear spatial visualization of LPLs in complex biological tissues. A series of lysophosphatidylcholines (LPCs) was detected using positive ion detection mode, and their concentration differences and spatial distributions were clearly visualized in different parts of zebrafish tissue. The method is rapid, simple, and efficient, being a desirable way to understand the spatial distribution of LPLs in biosome.

Luteolin attenuates imiquimod-induced psoriasis-like skin lesions in BALB/c mice via suppression of inflammation response.

Psoriasis is considered as a common chronic immune-mediated skin disorder characterized by abnormal keratinocyte proliferation. Luteolin, an anti-inflammatory natural flavonoid with well-accepted inhibition effect against keratinocyte proliferation, was hypothesized to have a potential therapeutic effect for psoriasis. In this paper, we investigated the relieving effect of luteolin against imiquimod-induced psoriasis-like lesions on BALB/c mice and its possible anti-inflammatory mechanisms in lipopolysaccharide-stimulated macrophages (RAW264.7 cells). We found that luteolin ameliorated psoriasis-like skin lesions, suppressed the cutaneous infiltration of macrophages, T cells and neutrophils, and downregulated the expression of cytokines like IL-6, IL-1ß, TNF-α, IL-17A and IL-23 in both skin lesions and eyeball blood of model mice. In vitro, we observed luteolin significantly suppressed the levels of psoriasis-related pro-inflammatory cytokines, such as IL-17A, IL-6, TNF-α and IL-23, and inflammatory mediators like nitric oxide NO, inducible NOS, COX-2 in RAW264.7 cells. The anti-inflammatory activity was accomplished by inhibiting NF-κB expression and activation. This study demonstrates luteolin is effective in alleviating psoriasis-like skin lesions and downregulating inflammatory response via NF-κB pathway, suggesting luteolin as a potential molecule for further therapeutic research of inflammation-related skin diseases like psoriasis.

Analysis of trace malachite green, crystal violet, and their metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry.

Malachite green (MG) and crystal violet (CV) are the typical triphenylmethane dyes, which are recalcitrant molecules exerting mutagenic and carcinogenic effects on living organisms. Characterization of the residues of MG, CV, and their metabolites in biological organisms is of importance, especially for in vivo and in situ characterization. In this study, a method for determination of trace MG, CV, and their leuco metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) was developed. A microscale solid-phase microextraction (SPME) probe was developed and used for extraction and enrichment of trace MG, CV, and their leuco metabolites in zebrafish after exposure. After that, the loaded SPME probe was directly employed for nanoESI-MS analysis under ambient and open-air conditions. Under the optimum conditions, the method demonstrated good linearity, with correlation coefficient values (r2) no less than 0.9925. The limits of detection and quantification were 0.014-0.023 ng mL-1 and 0.046-0.077 ng mL-1, respectively. By using the proposed method, the bioaccumulation of MG and CV in zebrafish was investigated, and the distribution of MG, CV, and their leuco metabolites in different organs of zebrafish was studied. MG, CV, and their leuco metabolites were all found in zebrafish tissues including brain, muscle, heart, and kidney after exposure, with highest concentration in intestine followed in ovary.

A heterogeneous pore decoration strategy on a hydrophobic microporous polymer for high-coverage capture of metabolites.

Herein, a heterogeneous pore decoration strategy on a hydrophobic microporous polymer is presented. The smaller pores in the material were completely decorated with hydrophilic polydopamine while most of the larger ones survived after modification, leading to its hydrophobic-hydrophilic hybrid properties and high-coverage capture ability of metabolites.

Carbon dots based solid phase microextraction of 2-nitroaniline followed by fluorescence sensing for selective early screening and sensitive gas chromatography-mass spectrometry determination.

For decades, researches have been involving in improving and optimizing solid-phase microextraction (SPME) technique to fit the growing environmental analysis demand. But it is more significant now to introduce meaningful information into the analysis line instead of improving every step in SPME. Herein, we design a carbon dots (CDs) based SPME platform and report its unique application in selective early screening and sensitive detection of 2-nitroaniline. The fluorescence quenching state of the CDs fiber after extraction could be used to prejudge whether an environmental sample contains 2-nitroaniline exceeding the maximum permitted level (30 µg L-1) regulated by the Chinese Ministry of Environmental Protection or not. Photo-induced electron transfer (PET) is believed to be the primary quenching route that leads to electron transfer from the electron-rich CDs to the electron-deficient analyte. A preliminary in vivo study on Daphnia magna and a 2-nitroaniline assay in environmental water and biofluid samples suggested the safety and the excellent qualitative ability of the proposed platform. As a result, the innovative incorporation of fluorescence sensing and SPME not only achieves the information imbedding of environmental analysis system, but also combines the advantages of two techniques including early visual-screening, reusability (≥50 times), high specificity and excellent quantitative ability (detection limit: 0.011 µg L-1). We believe this study can open new avenues in the field of environmental toxicant monitoring.

Comparative Proteomic Analysis by iTRAQ Reveals that Plastid Pigment Metabolism Contributes to Leaf Color Changes in Tobacco (Nicotiana tabacum) during Curing.

Tobacco (Nicotiana tabacum), is a world's major non-food agricultural crop widely cultivated for its economic value. Among several color change associated biological processes, plastid pigment metabolism is of trivial importance in postharvest plant organs during curing and storage. However, the molecular mechanisms involved in carotenoid and chlorophyll metabolism, as well as color change in tobacco leaves during curing, need further elaboration. Here, proteomic analysis at different curing stages (0 h, 48 h, 72 h) was performed in tobacco cv. Bi'na1 with an aim to investigate the molecular mechanisms of pigment metabolism in tobacco leaves as revealed by the iTRAQ proteomic approach. Our results displayed significant differences in leaf color parameters and ultrastructural fingerprints that indicate an acceleration of chloroplast disintegration and promotion of pigment degradation in tobacco leaves due to curing. In total, 5931 proteins were identified, of which 923 (450 up-regulated, 452 down-regulated, and 21 common) differentially expressed proteins (DEPs) were obtained from tobacco leaves. To elucidate the molecular mechanisms of pigment metabolism and color change, 19 DEPs involved in carotenoid metabolism and 12 DEPs related to chlorophyll metabolism were screened. The results exhibited the complex regulation of DEPs in carotenoid metabolism, a negative regulation in chlorophyll biosynthesis, and a positive regulation in chlorophyll breakdown, which delayed the degradation of xanthophylls and accelerated the breakdown of chlorophylls, promoting the formation of yellow color during curing. Particularly, the up-regulation of the chlorophyllase-1-like isoform X2 was the key protein regulatory mechanism responsible for chlorophyll metabolism and color change. The expression pattern of 8 genes was consistent with the iTRAQ data. These results not only provide new insights into pigment metabolism and color change underlying the postharvest physiological regulatory networks in plants, but also a broader perspective, which prompts us to pay attention to further screen key proteins in tobacco leaves during curing.

Characterization of Volatile Compounds in Four Different Rhododendron Flowers by GC×GC-QTOFMS.

Volatile compounds in flowers of Rhododendron delavayi, R. agastum, R. annae, and R. irroratum were analyzed using comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC) coupled with high-resolution quadrupole time-of-flight mass spectrometry (QTOFMS). A significantly increased number of compounds was separated by GC×GC compared to conventional one-dimensional GC (1DGC), allowing more comprehensive understanding of the volatile composition of Rhododendron flowers. In total, 129 volatile compounds were detected and quantified. Among them, hexanal, limonene, benzeneacetaldehyde, 2-nonen-1-ol, phenylethyl alcohol, citronellal, isopulegol, 3,5-dimethoxytoluene, and pyridine are the main compounds with different content levels in all flower samples. 1,2,3-trimethoxy-5-methyl-benzene exhibits significantly higher content in R. irroratum compared to in the other three species, while isopulegol is only found in R. irroratum and R. agastum.

A microscale solid-phase microextraction probe for the in situ analysis of perfluoroalkyl substances and lipids in biological tissues using mass spectrometry.

The simultaneous analysis of perfluoroalkyl substances (PFASs) and lipids in biological tissues is of importance, especially for in situ and microscale analysis, because it provides significant information to understand the relevance of content, composition, and distribution of lipids to the bioaccumulation of PFASs as well as lipid metabolism affected by the biotoxicity of PFASs. In this study, we report the development of a novel ambient mass spectrometry method for the rapid, in situ, and microscale analysis of PFASs and lipids simultaneously in biological tissues for the investigation of their biological correlation. A microscale solid-phase microextraction (SPME) probe with a probe-end diameter of several-µm was employed for in situ and microscale sampling of biological tissues after PFAS exposure. The SPME probe showed a desirable capacity for the enrichment of PFASs and lipid species simultaneously. After sampling and extraction, the loaded SPME probe was directly applied for nanoESI-MS analysis under ambient and open-air conditions. A high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated in the field-induced mode was introduced to record mass spectra using fast polarity switching between positive and negative ion detection. Most of the lipid species were recorded in the positive ion mass spectrum, and PFASs were recorded in the negative ion mass spectrum. By using the developed method, the in situ analysis of PFASs and lipids in the muscle, brain, heart, kidney, liver, and intestine of zebrafish was realized. In addition, simultaneously imaging PFASs and lipids in individual Daphnia magna was successfully achieved for the investigation of their biological correlation.

Analysis of volatile components in Dalbergia cochinchinensis Pierre by a comprehensive two-dimensional gas chromatography with mass spectrometry method using a solid-state modulator.

Volatile components in Dalbergia cochinchinensis Pierre were analysed using a comprehensive two-dimensional gas chromatography with mass spectrometry method featuring a new solid-state modulator. Compared to one-dimensional gas chromatography, the number of detected peaks were significantly increased. A total of 45 major compounds were identified in this study and the forward and reverse match factors of these compounds were both above 800. The results showed that the volatile components in Dalbergia cochinchinensis Pierre were primarily aldehyde and ketone compounds such as benzaldehyde, cinnamaldehyde, 4-chromanone, 1-(2-hydroxyphenyl)ethanone and acetophenone. In addition, a semi-quantitative analysis was conducted to determine the contents of the detected compounds based on peak area percentage. Moreover, the repeatability of the comprehensive two-dimensional gas chromatography-mass spectrometry analysis in this study was quite satisfactory with relative standard deviations less than 12.7% for intraday and 17.3% for interday measurements.

Free amino acids, biogenic amines, and ammonium profiling in tobacco from different geographical origins using microwave-assisted extraction followed by ultra high performance liquid chromatography.

This work describes a rapid, stable, and accurate method for determining the free amino acids, biogenic amines, and ammonium in tobacco. The target analytes were extracted with microwave-assisted extraction and then derivatized with diethyl ethoxymethylenemalonate, followed by ultra high performance liquid chromatography analysis. The experimental design used to optimize the microwave-assisted extraction conditions showed that the optimal extraction time was 10 min with a temperature of 60°C. The stability of aminoenone derivatives was improved by keeping the pH near 9.0, and there was no obvious degradation during the 80°C heating and room temperature storage. Under optimal conditions, this method showed good linearity (R2 > 0.999) and sensitivity (limits of detection 0.010-0.081 µg/mL). The extraction recoveries were between 88.4 and 106.5%, while the repeatability and reproducibility ranged from 0.48 to 5.12% and from 1.56 to 6.52%, respectively. The newly developed method was employed to analyze the tobacco from different geographical origins. Principal component analysis showed that four geographical origins of tobacco could be clearly distinguished and that each had their characteristic components. The proposed method also showed great potential for further investigations on nitrogen metabolism in plants.

Low-temperature derivatization followed by vortex-assisted liquid-liquid microextraction for the analysis of polyamines in Nicotiana Tabacum.

Polyamines are ubiquitous polycationic molecules that play a key role in many biological processes such as nucleic acid metabolism, protein synthesis, cell growth, and nicotine synthesis precursors. This work describes a rapid, sensitive, convenient, green, and cost-effective method for the determination of polyamines in Nicotiana tabacum by ultra high performance liquid chromatography with photodiode array detection. The analytes were derivatized with 3,5-dinitrobenzoyl chloride at low temperature (about 4°C) and then extracted with vortex-assisted liquid-liquid microextraction. The experimental designs based on quarter-fractional factorial design and Doehlert design were used to screen and optimize the important factors in microextraction process. Under the optimal conditions, the method was linear over 0.05-8.00 µg/mL with an r(2) ≥ 0.992 and exhibited good repeatability and reproducibility less than 6.0 and 6.9%, respectively. The limit of detection ranged between 0.013 and 0.029 µg/g. The newly developed method was successfully employed to analyze different leaf samples of Nicotiana tabacum, among which the polyamines contents were found to be very different. Moreover, tyramine, 1,3-diaminopropane, homospermidine, and canavalmine were tentatively identified with the electrospray ionization quadrupole time-of-flight mass spectrometry. To our knowledge, this is the first report of identification of canavalmine in Nicotiana Tabacum.